1. Field of the Invention
This invention relates generally to sensing instruments and methods for measuring the concentration of an analyte in a medium and, more particularly, to a device and method for measuring such concentrations by measuring the emission time delay during irradiation of a targeted sample surrounded by the analyte. Specifically, the present invention relates to a device and method for measuring exponential time constants, phase shifts, time delays and parameters derivable therefrom caused by irradiation of a targeted sample utilizing digital signal processing and especially luminescence quenching systems, phase shifts through networks, and time delays of photon migration through media.
2. Description of the Prior Art
Dynamic phase modulation, quenched luminescence sensors are well known. Instruments of this type have been, for example, developed or proposed for use in hospitals to monitor the concentration of gases such as oxygen, ionized hydrogen and carbon dioxide within the blood of patients. The particular substance of interest, for example oxygen, is known as the analyte.
As is known in the art, luminescence materials absorb energy and are driven from their ground state energy level to an excited state energy level in response to the application of energy from an electromagnetic radiation source such as light. These materials are unstable in their excited states, and they luminesce or give off excess energy as they return to their ground state. For example, the short wavelength ultraviolet light of black light stimulates dyes in a colored fabric to emit longer wavelengths, such as blue, green or red, and thus fluoresce. For the purposes of the present disclosure, the term “luminescence” as used herein is a general term which describes both fluorescence and phosphorescence, for all three terms are frequently used interchangeably in the art. The distinction and overlap of the terms is obvious to one skilled in the art.
In the presence of certain chemicals, many fluorescent materials are said to be quenched, i.e. the time constant of the fluorescence emission is altered by the effects of the surrounding chemicals. The degree of quenching of the fluorescence in turn can be related to the concentration of the quencher, which for example may be a chemical dissolved in water or mixed in air, such as oxygen in the blood of patients as explained above. There is a substantial amount of literature that describes fluorescent molecules that are selectively quenched by oxygen, carbon dioxide, glucose, pH, NH3, metal ions, temperature and other environmentally and medically important analytes. These analytes are relevant to applications such as monitoring drinking water quality, industrial process control, monitoring of human respiratory function, human blood analysis for critical care patients, and the like.
One of the obstacles to the commercialization of fluorescence sensing devices has been a lack of inexpensive yet accurate instrumentation for the measurement of changes in the fluorescent time constant. For example, U.S. Pat. Nos. 4,845,368, 5,257,202, 5,495,850, 5,515,864 and 5,626,134 all disclose devices for measuring analyte concentration levels based on fluorescence. However, these particular devices are generally expensive and complicated.
The fluorescence lifetime or time constant, τ is the amount of time it takes the fluorescence emission to decrease by a factor of 1/e or about 63% after termination of irradiation as disclosed in U.S. Pat. No. 4,716,363 by Dukes et al, in column 1, lines 37-41. This is common knowledge and is available in the literature reference, i.e. “Topics in Fluorescence Vol. 2—Principles”, ed. Joseph Lakowicz. If light modulated sinusoidally at a frequency, ƒ, is thus applied to the fluorescence sensor, the output is a sinusoidal emission of identical frequency, but having a phase shift and reduced amplitude with respect to the excitation signal. The equation governing the relationship between modulation frequency, ƒ, phase shift, θ, and the fluorescent time constant, τ, is as follows:                     τ        =                                                            tan                ⁢                                                                   ⁢                θ                                            2                ⁢                π                ⁢                                                                   ⁢                f                                      ⁢                                                   ⁢            or            ⁢                                                   ⁢            θ                    =                      arctan            ⁡                          (                              2                ⁢                π                ⁢                                                                   ⁢                f                ⁢                                                                   ⁢                τ                            )                                                          (                  Equation          ⁢                                           ⁢          1                )            orθ=arctan(2πƒτ)   (Equation 1) 
Thus, if we know the excitation modulation frequency and can measure the phase shift of the emission signal relative to the excitation signal, we can determine the fluorescence constant, τ, using the above Equation 1. In a fluorescence-based sensor, the fluorescence time constant is measured since this fluorescence time constant is altered by the presence of certain chemical species. Consequently, the concentration of chemical species can be determined by measuring the fluorescence time constant by measuring the phase shift associated therewith.
According to Equation 1, in order to measure the fluorescence time constant, one must know the excitation modulation frequency, ƒ, and the phase shift of the light through the fluorescence system. With these quantities, the fluorescence time constant can be calculated and then related to analyte concentration. There are several different known techniques for determining the excitation frequency and phase shift of a system with an unknown time constant. One manner of determining this is by exciting the sample with a fixed frequency signal and then measuring the phase shift that results, that is the sample excitation modulation frequency is maintained constant while the signal phase, which varies with analyte concentration, is measured. U.S. Pat. Nos. 5,317,162, 5,462,879, 5,485,530 and 5,504,337 all disclose such fixed frequency, variable phase techniques and devices. Of particular interest is an article by Venkatesh Vadde and Vivek Srinivas entitled, “A closed loop scheme for phase-sensitive fluorometry”, the American Institute of Physics, Rev. Sci. Instrum., Vol. 66, No. 7, July 1995, p. 3750.
Another principal way of conducting the above measurements is by exciting the sample with a modulation frequency that maintains a constant phase relationship between the excitation signal and the emission signal, that is the excitation frequency is varied in order to maintain a particular phase relationship. Such devices and techniques are known as phase-modulation, fluorescence-based sensing devices and are clearly illustrated in U.S. Pat. Nos. 4,840,485, 5,196,709, and 5,212,386, and in an article by Brett A. Feddersen, et al. entitled, “Digital parallel acquisition in frequency domain fluorimetry”, American Institute of Physics, Rev. Sci. Instrum., Vol. 60, No. 9, September 1989, p. 2929. Of particular interest in U.S. Pat. No. 4,716,363 by Dukes et al., which describes a feedback system that provides the modulation frequency required to give a constant phase shift of about 45°. The resulting frequency is then used to determine the analyte concentration which is a function of excited state lifetime.
U.S. Pat. No. 5,818,582 teaches the use of a DSP for fluorescence lifetime measurements, though not using quadrature signal comparison for determination of fluorescent sample phase shifts.
Despite the availability of the above-discussed techniques and sensing devices, there is a continuing need for improved fluorescence-based sensing instruments. In particular, there is a need for such devices which are useful for a broad range of applications involving exponential decay and time delay measurements, which are made from inexpensive components, and which present measurements in real time without the need for off-line signal processing as is the case of the patents to Federson, Gratton and others. A major detriment to many of the devices presently available is that they are very expensive to acquire and maintain. Moreover, analog systems of the present art are subject to drift and therefore unnecessary errors. Such systems should be, to the contrary, inexpensive, convenient to use and provide adequate sensitivity over an extended and continuous measurement range. The system of the Dukes patent emphasizes optimal sensitivity over a wide measurement range, but in so doing, requires very complex and expensive system components. To the contrary, optimal sensitivity can be sacrificed for sub-optimal, adequate sensitivity in order to achieve inexpensive, less complicated measurement techniques. In addition, the measurement approach of such devices should be susceptible to convenient and precise readout.